Process for obtaining insulin precursors having correctly bonded cystine bridges

ABSTRACT

The present invention relates to an improved process for obtaining a precursor of an insulin or insulin derivative having correctly bonded cystine bridges in the presence of cysteine or cysteine hydrochloride and chaotropic auxiliary.

This is a division of U.S. patent application Ser. No. 09/134,836, filedon Aug. 19, 1998 now U.S. Pat. No. 5,986,048.

BACKGROUND OF THE INVENTION

The present invention relates to an improved process for obtaining aprecursor of insulin or an insulin derivative having correctly bondedcystine bridges in the presence of cysteine or cysteine hydrochlorideand a chaotropic auxiliary.

Human insulin is a protein with two amino acid chains together having 51amino acid residues. Six cysteine residues are found in the two aminoacid chains, each two cysteine residues being bonded to one another viaa disulfide bridge. In biologically active human insulin, the A and Bchains are bonded to one another via two cystine bridges, and a furthercystine bridge is found in the A chain. Within a human insulin molecule,looked at statistically, there are 15 possibilities for the formation ofdisulfide bridges. In biologically active human insulin, only one of the15 possibilities is found. The following cysteine residues are linked toone another in human insulin:

A 6-A 11

A 7-B 7

A 20-B 19

The letters A and B represent the respective insulin amino acid chainand the numbers represent the position of the amino acid residue, whichis counted from the amino to the carboxyl end of the respective aminoacid chain. Disulfide bridges can also be formed between two humaninsulin molecules such that an incalculable number of differentdisulfide bridges can easily result.

A known process for the preparation of human insulin is based on the useof human proinsulin. Human proinsulin is a protein having a linear aminoacid chain of 86 amino acid residues, the A and B chains of the humaninsulin being bonded to one another via a C peptide having 35 amino acidresidues. The formation of the disulfide bridges found in human insulintakes place via an intermediate, the cysteine residues of the humaninsulin being provided with a sulfur protective group, e.g. anS-sulfonate (—S—SO3—) group (EP 0 037 255). A process for obtainingproinsulin having correctly bonded cystine bridges is additionally known(Biochemistry, 60, (1968), pages 622 to 629), which starts fromproinsulin obtained from porcine pancreas, in which the cysteineresidues are present as thiol residues (—SH). The term “correctly bondedcystine bridges” is understood as meaning the disulfide bridges whichare found in biologically active insulin from mammals.

Recombinant DNA processes allow precursors of insulin or insulinderivatives, in particular human proinsulin or proinsulin which has anamino acid sequence and/or amino acid chain length differing from humaninsulin, to be prepared in microorganisms. The proinsulins prepared fromgenetically modified Escherichia coli cells do not have any correctlybonded cystine bridges. A process for obtaining human insulin using E.coli (EP 0 055 945) is based on the following process steps:

Fermentation of the microorganisms—cell disruption—isolation of thefusion protein—cyanogen halide cleavage of the fusion protein—isolationof the cleavage product having the proinsulin sequence—protection of thecystine residues of proinsulin by S-sulfonate groups—chromatographicpurification of the S-sulfonate—formation of the correctly bondedcystine bridges—desalting of the proinsulin—chromatographic purificationof the proinsulin having correctly bonded cystine bridges—concentrationof the proinsulin solution—chromatographic purification of theconcentrated proinsulin solution—enzymatic cleavage of the proinsulin toobtain human insulin—chromatographic purification of the resulting humaninsulin.

Disadvantages of this process are the number of process steps and thelosses in the purification steps, which lead to a low yield of insulin.From the stage of the isolated fusion protein via cyanogen halidecleavage, sulfitolysis and purification of the proinsulin, a loss of upto 40% of the proinsulin product has to be expected (EP 0 055 945).Similarly, high losses can occur in the course of the subsequentrequired to obtain the final product.

Yield increases in the preparation of human insulin or insulinderivatives by recombinant DNA means can be achieved if the number ofprocess steps necessary can be significantly reduced.

EP 0 600 372 A1 (or U.S. Pat. No. 5,473,049) and EP 0 668 292 A2disclose an improved process for obtaining insulin or insulinderivatives, in which the insulin precursor or precursor of the insulinderivative whose cystine bridges are not present in correctly linkedform is reacted in the presence of a mercaptan, for example cysteine,and of at least one chaotropic auxiliary, for example urea or guanidinehydrochloride, to give an insulin precursor or precursor of the insulinderivative having correctly bonded cystine bridges. In the knownprocess, these proteins are first dissolved in a very low concentrationin aqueous solutions of a chaotropic auxiliary or of mixtures of variouschaotropic auxiliaries. The protein mixture is then mixed with anaqueous mercaptan solution.

Surprisingly, it has now been found that the yields of correctly foldedprecursors of insulins or insulin derivatives can be increased and thereaction times for the folding process can be reduced by delaying thedissolution of the precursor in a first step by means of the chaotropicauxiliary. Instead, a mercaptan, such as cysteine or cysteinehydrochloride, is introduced into the aqueous suspension of theprecursor in the first step, followed by dissolution of the precursor ina subsequent step by introducing a chaotropic auxiliary, and finallybringing about the correct folding of the precursor by dilution of themixture to a preferred cysteine or cysteine hydrochloride concentrationwith an appropriate amount of water.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a process for obtaining aprecursor of insulin or an insulin derivative having correctly bondedcystine bridges in the presence of cysteine or cysteine hydrochlorideand of a chaotropic auxiliary, which comprises successively carrying outthe following steps:

(a) mixing an aqueous suspension of the precursor of insulin or aninsulin derivative with an amount of cysteine or cysteine hydrochloridewhich results in approximately 1 to 15 SH residues of the cysteine orcysteine hydrochloride per cysteine residue of the precursor,

(b) introducing the cysteine- or cysteine hydrochloride-containingsuspension of the precursor into an approximately 4 to 9 molar solutionof the chaotropic auxiliary at a pH of approximately 8 to 11.5 and atemperature of approximately 15° C. to 55° C., keeping the mixtureobtained at this temperature for approximately 10 to 60 minutes; and

(c) introducing the mixture at a pH of approximately 8 to 11.5 and atemperature of approximately 5° C. to 30° C. into an amount of waterwhich results in a dilution of the concentration of the cysteine or ofthe cysteine hydrochloride in the mixture to approximately 1 to 5 mM andof the chaotropic auxiliary to approximately 0.2 to 1.0 M.

DETAILED DESCRIPTION

Preferably, the process is one wherein in step (a) the amount ofcysteine or cysteine hydrochloride corresponds to an amount whichresults in approximately 1 to 6 SH residues of the cysteine or cysteinehydrochloride per cysteine residue of the precursor,

in step (b) the cysteine- or cysteine hydrochloride-containingsuspension of the precursor is introduced into an approximately 4 to 9molar solution of the chaotropic auxiliary at a pH of approximately 8 to11 and a temperature of approximately 30° C. to 45° C. and the mixtureobtained is kept for approximately 20 to 40 minutes at this temperature;and

in step (c) the mixture is introduced at a pH of approximately 8 to 11and at a temperature of approximately 15° C. to 20° C. into an amount ofwater which results in a dilution of the concentration of the cysteineor of the cysteine hydrochloride in the mixture to approximately 1 to 5mM and a concentration of the chaotropic auxiliary of approximately 0.2to 1.0 M.

Chaotropic auxiliaries are compounds which break hydrogen bridges inaqueous solution, for example ammonium sulfate, guanidine hydrochloride,ethylene carbonate, thiocyanate, dimethyl sulfoxide and urea.

In the process of this invention, the chaotropic auxiliary employed ispreferably guanidine, guanidine hydrochloride or particularlypreferably, urea.

The concentration of the chaotropic auxiliary in step (b) of the processof this invention is preferably 7.0 to 9M, the temperature in step (b)is preferably 40° C. and the pH in step (b) is preferably 10 to 11.

In the process of this invention, the pH in step (c) is preferably 10 to11. In step (c) of the process of this invention, the amount of waterinto which the mixture is introduced is preferably selected such that itresults in a dilution of the cysteine or cysteine hydrochlorideconcentration in the mixture to approximately 2.5 to 3 mM and aconcentration of the chaotropic auxiliary of approximately 0.5 M.

Particularly preferably, the process according to the invention is onewherein the concentration of the chaotropic auxiliary in step (b) isapproximately 8 M, the temperature in step (b) is approximately 40° C.,the pH in step (b) is approximately 10.6, the pH in step (c) isapproximately 10.6 and in step (c) the amount of water results in adilution of the concentration of the cysteine or of the cysteinehydrochloride in the mixture to approximately 2.5 to 3 mM and aconcentration of the chaotropic auxiliary of approximately 0.5 M.

The result of the process of this invention is a precursor of insulin oran insulin derivative, in particular a proinsulin, whose cystine bridgesare correctly bonded.

Insulin derivatives are derivatives of naturally occurring insulins,namely human insulin (see SEQ ID NO:1=A chain of human insulin; see SEQID NO:2=B chain of human insulin, sequence listing) or animal insulins,which differ by substitution of at least one naturally occurring aminoacid residue and/or addition of at least one amino acid residue and/ororganic residue of the corresponding, otherwise identical, naturallyoccurring insulin.

From the precursor of the insulin or insulin derivative having correctlybonded cystine bridges obtained with the aid of this invention, it isfinally possible in combination with the process described in EP 0 600372 A1 (or U.S. Pat. No. 5,473,049) or in EP 0 668 292 A2, to prepare aninsulin or an insulin derivative having correctly bonded cystine bridgesby enzymatic cleavage by means of trypsin or a tripsin-like enzyme and,if appropriate, additionally by means of carboxypeptidase B andsubsequent purification on an adsorber resin.

The insulin or insulin derivative which can be prepared from theprecursor can preferably be described by formula I

in which

Y is a genetically encodable amino acid residue;

z is

a) an amino acid residue from the group consisting of His, Arg and Lys,

b) a peptide having 2 or 3 amino acid residues, comprising the aminoacid residue Arg or Lys at the carboxyl end of the peptide,

c) a peptide having 2-35 genetically encodable amino acids, comprising 1to 5 histidine residues, or

d) OH;

R¹ is a phenylalanine residue (Phe) or a covalent bond,

R³ is a genetically encodable amino acid residue,

and where the radicals A2-A20 of the amino acid sequence of the A chainof human insulin (not shown for the simplification of the formula I)correspond to animal insulin or an insulin derivative and the radicalsB2-B29 of the amino acid sequence of the B chain of human insulin (notshown for the simplification of the formula I) correspond to animalinsulin or an insulin derivative.

The amino acid sequence of peptides and proteins is indicated from theN-terminal end of the amino acid chain onward. The details in formula Iin brackets, e.g. A6, A20, B1, B7 or B19, correspond to the position ofamino acid residues in the A or B chains of the insulin.

The term “genetically encodable amino acid residue” represents the aminoacids Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met,Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine.

The terms “residues A2-A20” and “residues B2-B29” of “animal insulin”are understood as meaning, for example, the amino acid sequences ofinsulin from cattle, pigs or chickens. The terms “residues A2-A20” and“B2-B29” of insulin derivatives represent the corresponding amino acidsequences of human insulin which are formed by the replacement of aminoacids by other genetically encodable amino acids.

The A chain of human insulin has the following sequence (SEQ ID NO:1):

Gly Ile Val Glu Gin Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu AsnTyr Cys Asn.

The B chain of human insulin has the following sequence (SEQ ID NO:2):

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu ValCys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr.

In human insulin, R³ in formula I is asparagine (Asn), R¹ isphenylalanine (Phe), Y is threonine (Thr) and Z is OH.

The process according to the present invention is particularly suitablefor obtaining a precursor of insulin or an insulin derivative having theformula II, whose cystine bridges (not shown in formula II) arecorrectly folded,

R²—R¹—(B2-B29)—Y—X-Gly-(A2-A20)—R³  (II),

in which

R² is

a) a hydrogen atom,

b) an amino acid residue from the group consisting of lysine (Lys) andarginine (Arg), or

c) a peptide having 2 to 45 amino acid residues, comprising the aminoacid residue lysine (Lys) or arginine (Arg) at the carboxyl end of thepeptide;

R¹ is a phenylalanine residue (Phe) or a covalent bond;

(B2-B29) are the amino acid residues in the positions B2 to B29 of the Bchain of human insulin, animal insulin or an insulin derivative which isoptionally varied in one or more of these positions;

Y is a genetically encodable amino acid residue;

X is

a) an amino acid residue from the group consisting of lysine (Lys) andarginine (Arg),

b) a peptide having 2 to 35 amino acid residues, comprising the aminoacid residue lysine (Lys) or arginine (Arg) at the N-terminal end and atthe carboxyl end of the peptide, or

c) a peptide having 2 to 35 genetically encodable amino acids,comprising 1 to 5 histidine residues;

(A2-A20) are the amino acid residues in the positions A2 to A20 of the Achain of human insulin, animal insulin or an insulin derivative which isoptionally varied in one or more of these positions; and

R³ is a genetically encodable amino acid residue.

1. In a first preferred embodiment, in formula II:

R² is

a) a hydrogen atom, or

b) a peptide having 2 to 25 amino acid residues, comprising the aminoacid residue arginine (Arg) at the carboxyl end of the peptide;

R¹ is a phenylalanine residue (Phe);

(B2-B29) are the amino acid residues in the positions B2 to B29 of the Bchain of human insulin;

Y is an amino acid residue from the group consisting of alanine (Ala),threonine (Thr) and serine (Ser);

X is the amino acid residue arginine (Arg) or a peptide having the aminoacid sequence of the C chain of human insulin;

(A2-A20) are the amino acid residues in the positions A2 to A20 of the Achain of human insulin; and

R³ is an amino acid residue from the group consisting of asparagine(Asn), serine (Ser) and glycine (Gly)

The C chain of human insulin has the following sequence (SEQ ID NO:3):

Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly ProGly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg.

2. In a second preferred embodiment, in formula II:

R² is

a) a hydrogen atom, or

b) a peptide having 2 to 15 amino acid residues, at whose carboxyl endis found an arginine residue (Arg);

R¹ is a phenylalanine residue (Phe);

(B2-B29) are the amino acid residues in the positions B2 to B29 of the Bchain of human insulin;

Y is a threonine residue (Thr);

X is the amino acid residue arginine (Arg) or a peptide having 2 to 35amino acid residues, where at the beginning and at the end of thepeptide there are two basic amino acid residues, in particular arginine(Arg) and/or lysine (Lys);

(A2-A20) are the amino acid residues in the positions A2 to A20 of the Achain of human insulin; and

R³ is the amino acid residue asparagine (Asn) or glycine(Gly).

The residue Z of the insulin or of the insulin derivative of the formulaI is, as a rule, part of the amino acid sequence of X of the precursorof the formula II and results from the activity of the proteases such astrypsin, trypsin-like enzyme or carboxypeptidase B, therefor The radicalR³ is the amino acid residue which is in position A21 of the A chain ofinsulin. The radical Y is the amino acid residue which is in positionB30 of the B chain of insulin.

Trypsin or trypsin-like enzymes are proteases which cleave amino acidchains at the arginine or lysine residue. Carboxypeptidase B is anxoprotease which removes basic amino acid residues such as Arg or Lyswhich are at the carboxy-terminal end of amino acid chains. (Kemmler etal., J. Biol. Chem. 246, pages 6786-6791).

From the precursor described above as the first preferred embodiment, itis possible, for example, to obtain an insulin or insulin derivative ofthe formula I having correctly linked cystine bridges, where Y, R¹, R²,R³, A2-A20 and B2-B29 have the precursor described above as a firstpreferred embodiment, and Z is an argine residue (Arg), a peptideresidue Arg-Arg or —OH.

From the precursor precursor described above as the second preferredembodiment, it is possible, for example, to obtain an insulin or insulinderivative of the formula I having correctly linked cystine bridges,where Y, R¹, R², R³, A2-A20 and B2-B29 have the meaning precursordescribed above as a second preferred embodiment,and Z is an arginineresidue (Arg), a peptide residue Arg-Arg or Lys-Lys or —OH.

The precursor of the formula II can be formed in microorganisms with theaid of a large number of genetic constructs (EP 0 489 780, EP 0 347 781,EP 0 453 969). The genetic constructs are expressed in microorganismssuch as Escherichia coli or Streptomycetes during fermentation. Theproteins formed are deposited in the interior of the microorganisms (EP0 489 780) or secreted into the fermentation solution.

For the process according to the invention, precursors of insulins or ofinsulin derivatives of the formula II can be employed that are stillcontaminated with a large number of proteins which originate from thefermentation solution and from the microorganisms directly after thecell disruption. The precursors of the formula II, however, can also beemployed in prepurified form, for example after precipitation orchromatographic purification.

EXAMPLE 1 Comparison Example, Prior Art

By fermentation of genetically modified Escherichia coli cells (EP 0 489780), a fusion protein having the following amino acid sequence isprepared.

Proinsulin sequence 1 (SEQ ID NO:4):

Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln His Leu Cys GlySer His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe TyrThr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu LeuGly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser LeuGln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr GlnLeu Glu Asn Tyr Cys Asn

Proinsulin sequence 1 corresponds to the formula II, in this formula

X is C-peptide from human insulin (SEQ ID NO:3);

Y is Thr (B30);

R¹ is Phe(B1);

R² is a peptide having 10 amino acid residues;

R³ is Asn (A21); and

A2-A20 is the amino acid sequence of the A chain of human insulin (aminoacid residues 2 to 20) and B2-B29 is the amino acid sequence of the Bchain of human insulin (amino acid residues 2 to 29).

The expressed fusion protein having the proinsulin sequence 1 collectsin the E. coli cells and forms inclusion bodies. After completion of thefermentation, the cells are separated off by centrifugation anddisrupted by customary high-pressure homogenization. The fusion proteininclusion bodies released are isolated by centrifugation.

20 kg of the isolated fusion protein inclusion bodies (based on drymatter after freeze drying; the proportion of the insulin-containingfusion protein is determined with the aid of HPLC—it is 50%) having theproinsulin sequence 1 are dissolved in 550 liters of an 8 M ureasolution at pH 10.6. If appropriate, after centrifugation of smallamounts of substances causing turbidity, the clear solution is stirredinto 9000 liters of an aqueous cysteine solution (5 kg of cysteinehydrochloride hydrate) at a pH of 10.6 and a temperature of 4° C.

After completion of the folding reaction after about 24 h, the contentof proinsulin sequence 1 having correctly bonded cystine bridges in thereaction batch is determined with the aid of analytical HPLC as 3.0 kg,corresponding to a conversion of 30%.

The 9500 liters of solution is adjusted to a pH of 5.0 using 1N HCl andseparated. A pH of 9 is then set by addition of 1N sodium hydroxidesolution. 3 g of trypsin are introduced into the solution. 1.25 kg of aninsulin having 2 carboxy-terminal arginine residues results according toHPLC measurement.

After cleavage using carboxypeptidase B, human insulin results, which isadditionally purified with the aid of chromatographic methods.

Human insulin corresponds to the formula I, in this formula

Y is Thr (B30);

Z is OH;

R¹ is Phe (B1);

R³ is Asn (A21); and

A2-A20 is the amino acid sequence of the A chain of human insulin (aminoacid residues 2 to 20) and B2-B29 is the amino acid sequence of the Bchain of human insulin (amino acid residues 2 to 29).

Human insulin 2 consists of the SEQ ID NO:1 and SEQ ID NO:2, which areconnected to one another via correctly bonded cystine bridges.

As described in EP 0 668 292, the solution is concentrated and purifiedby means of adsorber resin. The eluate, which contains insulin 2, canimmediately be further purified on a chromatographic column afterdilution with water and pH adjustment.

HPLC analysis

0.5 g of protein is dissolved for 2 min in 40 ml of a solution of 6 Mguanidine hydrochloride, 50 mM tris, pH 8.5, 5 mM ethylenediaminetetraacetate (EDTA), 1% 2-mercaptoefhanol, 10 mM dithiothreitol at 95°C. and then centrifuged at 14000 g for 20 min. 0.02 ml of the clearsupernatant is applied to a high-pressure liquid chromatography column.

Column: ®Nucleogel RP 300-5/46 (Macherey & Nagel, Aachen, Germany)

Gradient: Buffer A: 0.1% trifluoroacetic acid (TFA) Buffer B: 0.09% TFAin acetonitrile

Temperature: 55° C.

Total run time: 40 min

The gradient is distinguished by the following amounts of buffer B afterthe corresponding run times:

10 min 25%, 12 min 60%, 13 min 90%, 15 min 100%.

Flow rate: 1 ml/min

Detection: 215 nm

Retention time of insulin: approximately 19 min

EXAMPLE 2 Process According to the Present Invention

By fermentation of genetically modified Escherichia coli cells (EP 0 489780), a fusion protein having the amino acid sequence shown in Example 1is prepared (proinsulin sequence 1; SEQ ID NO:4).

The expressed fusion protein having the proinsulin sequence 1 collectsin the E. coli cells and forms inclusion bodies. After completion of thefermentation, the cells are separated off by centrifugation anddisrupted by customary high-pressure homogenization. The released fusionprotein inclusion bodies are isolated by centrifugation.

5 kg of cysteine hydrochloride hydrate are added to the aqueous fusionprotein suspension, which contains 40 kg of fusion protein (determinedby freeze drying of an aliquot).

The suspension (the proportion of the insulin-containing fusion proteinis determined with the aid of HPLC—it is 50%) having the proinsulinsequence 1 is dissolved at 40° C. in 550 liters of an 8 M urea solutionat pH 10.2. The clear solution is stirred into 9000 liters of water at apH of 10.6 and a temperature of 15° C.

After completion of the folding reaction after about 5 hours, thecontent of proinsulin sequence 1 having correctly bonded cystine bridgesin the reaction batch is determined with the aid of analytical HPLC as10.0 kg, corresponding to a conversion of 50%.

The 9500 liters of solution is adjusted to a pH of 5.0 using 1N HCl andseparated. A pH of 9 is then set by addition of 1N sodium hydroxidesolution. 10 g of trypsin are introduced into the solution. 4 kg of aninsulin having 2 carboxy-terminal arginine residues result. Aftercleavage using carboxypeptidase B, human insulin (SEQ ID NO:1 and SEQ IDNO:2 having correctly bonded cystine bridges) results.

The solution is concentrated and purified by means of adsorber resin.The eluate which contains human insulin can immediately be purifiedfurther on a chromatography column after dilution with water and pHadjustment.

EXAMPLE 3 Comparison Example, Prior Art

By fermentation of genetically modified Escherichia coli cells (EP 0 489780), a fusion protein having the following amino acid sequence isprepared.

Proinsulin sequence 2 (SEQ ID NO:5)

Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln His Leu Cys GlySer His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe TyrThr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu LeuGly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser LeuGln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr GlnLeu Glu Asn Tyr Cys Gly

Proinsulin sequence 2 corresponds to the formula II, in this formula

X is C-peptide of human insulin (SEQ ID NO:3);

Y is Thr (B30);

R¹ is Phe (B1);

R² is a peptide having 10 amino acid residues;

R³ is Gly (A21); and

A2-A20 is the amino acid sequence of the A chain of human insulin (aminoacid residues 2 to 20) and B2-B29 is the amino acid sequence of the Bchain of human insulin (amino acid residues 2 to 29).

The expressed fusion protein having the proinsulin sequence 2 collectsin the E. coli cells and forms inclusion bodies. After completion of thefermentation, the cells are separated off by centrifugation anddisrupted by customary high-pressure homogenization. The released fusionprotein inclusion bodies are isolated by centrifugation.

20 kg of the isolated fusion protein inclusion bodies (based on drymatter after freeze drying; the proportion of the insulin-containingfusion protein is determined with the aid of HPLC—it is 50%.) having theproinsulin sequence 2 are dissolved at 20° C. in 550 liters of an 8 Murea solution at pH 10.6. The clear solution is stirred into 9000 litersof an aqueous cysteine solution (5 kg of cysteine hydrochloride hydrate)at a pH of 10.6 and a temperature of 4° C.

After completion of the folding reaction after about 24 hours, thecontent of proinsulin sequence 2 having correctly bonded cystine bridgesin the reaction batch is determined with the aid of analytical HPLC as3.0 kg, corresponding to a conversion of 30%.

The 9500 liters of solution is adjusted to a pH of 5.0 using 1N HCl andseparated. A pH of 9 is then set by addition of 1N sodium hydroxidesolution. 3 g of trypsin are introduced into the solution. 0.98 kg of aninsulin derivative having 2 carboxy-terminal arginine residues resultsaccording to HPLC measurement. This insulin derivative corresponds tothe formula I, where

Y is Thr (B30);

Z is Arg-Arg;

R¹ is Phe (B1);

R³ is Gly (A21); and

A2-A20 is the amino acid sequence of the A chain of human insulin (aminoacid residues 2 to 20) and B2-B29 is the amino acid sequence of the Bchain of human insulin (amino acid residues 2 to 29), and consists ofthe SEQ ID NO:6 and SEQ ID NO:7, which are connected to one another viacorrectly bonded cystine bridges.

The solution is concentrated and purified by means of adsorber resin.The eluate which contains the insulin derivative can immediately bepurified further on a chromatography column after dilution with waterand pH adjustment.

EXAMPLE 4 Process According to the Present Invention

By fermentation of genetically modified Escherichia coli cells (EP 0 489780), the fusion protein having the proinsulin sequence 2 (SEQ ID NO:5)is prepared according to Example 3.

The expressed fusion protein having the proinsulin sequence 2 collectsin the E. coli cells and forms inclusion bodies. After completion of thefermentation, the cells are separated off by centrifugation anddisrupted by customary high-pressure homogenization. The released fusionprotein inclusion bodies are isolated by centrifugation.

5 kg of cysteine hydrochloride hydrate are added to the aqueous fusionprotein suspension, which contains 40 kg of fusion protein (determinedby freeze drying of an aliquot).

The suspension (the proportion of the insulin-containing fusion proteinis determined with the aid of HPLC—it is 50%) having the proinsulinsequence 2 is dissolved at 40° C. in 550 liters of an 8 M urea solutionat pH 10.2. The clear solution is stirred into 9000 liters of water at apH of 10.6 and a temperature of 15° C.

After completion of the folding reaction after about 5 hours, thecontent of proinsulin sequence 1 having correctly bonded cystine bridgesin the reaction batch is determined with the aid of analytical HPLC as10.0 kg, corresponding to a conversion of 50%.

The 9500 liters of solution is adjusted to a pH of 5.0 using 1 N HCl andseparated. A pH of 9 is then set by addition of 1N sodium hydroxidesolution. 10 g of trypsin are introduced into the solution. 2.8 kg ofthe insulin derivative result (HPLC measurement), which consists of thesequences SEQ ID NO:6 and SEQ ID NO:7, which are linked to one anothervia correctly bonded cystine bridges.

The solution is concentrated by means of adsorber resin and purified.The eluate which contains the insulin derivative can immediately bepurified further on a chromatography column after dilution with waterand pH adjustment.

7 21 amino acids amino acid single linear protein Escherichia coliProtein 1..21 1 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu TyrGln Le 1 5 10 15 Glu Asn Tyr Cys Asn 20 30 amino acids amino acid singlelinear protein Escherichia coli Protein 1..30 2 Phe Val Asn Gln His LeuCys Gly Ser His Leu Val Glu Ala Leu Ty 1 5 10 15 Leu Val Cys Gly Glu ArgGly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 35 amino acids amino acidsingle linear protein Escherichia coli Protein 1..35 3 Arg Arg Glu AlaGlu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gl 1 5 10 15 Gly Pro Gly AlaGly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Le 20 25 30 Gln Lys Arg 3596 amino acids amino acid single linear protein Escherichia coli Protein1..96 4 Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gln His Le 15 10 15 Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Ar20 25 30 Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gl35 40 45 Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gl50 55 60 Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gl65 70 75 80 Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr CysAs 85 90 95 96 amino acids amino acid single linear protein Escherichiacoli Protein 1..96 5 Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val AsnGln His Le 1 5 10 15 Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val CysGly Glu Ar 20 25 30 Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala GluAsp Leu Gl 35 40 45 Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala GlySer Leu Gl 50 55 60 Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly IleVal Glu Gl 65 70 75 80 Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu GluAsn Tyr Cys Gl 85 90 95 32 amino acids amino acid single linear proteinEscherichia coli Protein 1..32 6 Phe Val Asn Gln His Leu Cys Gly Ser HisLeu Val Glu Ala Leu Ty 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe TyrThr Pro Lys Thr Arg Ar 20 25 30 21 amino acids amino acid single linearprotein Escherichia coli Protein 1..21 7 Gly Ile Val Glu Gln Cys Cys ThrSer Ile Cys Ser Leu Tyr Gln Le 1 5 10 15 Glu Asn Tyr Cys Gly 20

What is claimed is:
 1. A process for obtaining an insulin or an insulinderivative thereof having correctly bonded cystine bridges in thepresence of cysteine or cysteine hydrochloride and of a chaotropicauxiliary, which comprises the following steps: (a) mixing an aqueoussuspension of a precursor of insulin or insulin derivative with anamount of cysteine or cysteine hydrochloride which results inapproximately 1 to 15 SH residues of the cysteine or cysteinehydrochloride per cysteine residue of the precursor; (b) adding thecysteine- or cysteine hydrochloride-containing suspension of theprecursor into an approximately 4 to 9 molar solution of the chaotropicauxiliary at a pH of approximately 8 to 11.5 and a temperature ofapproximately 15° C. to 55° C., keeping the mixture obtained at thistemperature for approximately 10 to 60 minutes; (c) diluting thecysteine or the cysteine hydrochloride concentration to approximately 1to 5 mM and diluting the chaotropic auxilary concentration toapproximately 0.2 to 1.0 M in the mixture at a pH of approximately 8 to11.5 and a temperature of approximately 5° C. to 30° C.; (d) cleavingwith trypsin or a trypsin-like enzyme-the precursor of the insulin orinsulin derivative obtained by this process; and (e) purifying theinsulin or insulin derivative having correctly bonded cystine bridges.2. The process according to claim 1, wherein the chaotropic auxiliary isguanidine or guanidine hydrochloride.
 3. The process according to claim1, wherein the chaotropic auxiliary is urea.
 4. The process according toclaim 1, wherein the concentration of the chaotropic auxiliary in step(b) is approximately 7.0 to 9 M.
 5. The process according to claim 1,wherein the temperature in step (b) is approximately 40° C.
 6. Theprocess according to claim 1, wherein the pH in step (b) isapproximately 10 to
 11. 7. The process according to claim 1, wherein thepH in step (c) is approximately 10 to
 11. 8. The process according toclaim 1, wherein in step (c) the amount of water results in a dilutionof the concentration of the cysteine or of the cysteine hydrochloride inthe mixture to approximately 2.5 to 3 mM, and a concentration of thechaotropic auxiliary to approximately 0.5 M.
 9. The process according toclaim 4, wherein the concentration of the chaotropic auxiliary in step(b) is approximately 8 M, the temperature in step (b) is approximately40° C., the pH in step (b) is approximately 10.6, the pH in step (c) isapproximately 10.6 and in step (c) the amount of water results in adilution of the concentration of the cysteine or of the cysteinehydrochloride in the mixture to approximately 2.5 to 3 mM and aconcentration of the chaotropic auxiliary of approximately 0.5 M. 10.The process according to claim 1, wherein in step (a) the amount ofcysteine or cysteine hydrochloride corresponds to an amount whichresults in 1 to 6 SH residues of the cysteine or cysteine hydrochlorideper cysteine residue of the precursor, in step (b) the cysteine- orcysteine hydrochloride-containing suspension of the precursor is addedinto an approximately 4 to 9 molar solution of the chaotropic auxiliaryat a pH of approximately 8 to 11 and a temperature of approximately 30°C. to 45° C. and the mixture obtained is kept for approximately 20 to 40minutes at this temperature; and wherein the pH in step (c) isapproximately 8 to 11 and the temperature is approximately 15° C. to 20°C.
 11. The process according to claim 1, further comprising cleavage ofthe precursor of insulin or insulin derivative obtained in step (d) withcarboxypeptidase B or a carboxypeptidase-like enzyme.
 12. The processaccording to claim 1, wherein the insulin or insulin derivative has aformula I of

in which Y is a genetically encodable amino acid residue; Z is a) anamino acid residue from the group consisting of His, Arg and Lys, b) apeptide having 2 or 3 amino acid residues, comprising the amino acidresidue Arg or Lys at the carboxyl end of the peptide, c) a peptidehaving 2 to 35 genetically encodable amino acids, comprising 1 to 5histidine residues, or d) OH; R¹ is a phenylalanine residue (Phe) or acovalent bond, R³ is a genetically encodable amino acid residue, andwherein residues A2-A20 correspond to the amino acid sequence of the Achain of an animal insulin, or an insulin derivative thereof; andwherein residues B2-B29 correspond to the amino acid sequence of the Bchain of an animal insulin or an insulin derivative thereof.
 13. Theprocess according to claim 1, wherein in step (a) the precursor of theinsulin, or insulin derivative, has the formula IIR²—R¹—(B2-B29)—Y—X-Gly-(A2-A20)—R³  (II), wherein R² is a) a hydrogenatom, b) an amino acid residue from the group consisting of lysine (Lys)and arginine (Arg), or c) a peptide having 2 to 45 amino acid residues,comprising the amino acid residue lysine (Lys) or arginine (Arg) at thecarboxyl end of the peptide; R¹ is a phenylalanine residue (Phe) or acovalent bond; (B2-B29) are the amino acid residues in the positions B2to B29 of the B chain of an animal insulin or-an insulin derivativethereof; Y is a genetically encodable amino acid residue; X is a) anamino acid residue from the group consisting of lysine (Lys) andarginine (Arg), or b) a peptide having 2 to 35 amino acid residues,comprising the amino acid residue lysine (Lys) or arginine (Arg) at theN-terminal end at the carboxyl end of the peptide, or c) a peptidehaving 2 to 35 genetically encodable amino acids, comprising 1 to 5histidine residues; (A2-A20) are the amino acid residues in thepositions A2 to A20 of the A chain of human insulin, animal insulin oran insulin derivative thereof; and R³ is a genetically encodable aminoacid residue.
 14. The process of claim 12, wherein the insulin orinsulin derivative of formula I obtained corresponds to human insulin,wherein Y is Thr (B30); Z is OH; R¹ is Phe (B1); R³ is Asn (A21); andA2-A20 is the amino acid sequence of the A chain of human insulin (aminoacid residues 2 to 20) and B2-B29 is the amino acid sequence of the Bchain of human insulin (amino acid residues 2 to 29).
 15. The process ofclaim 12, wherein the insulin or insulin derivative of formula Iobtained corresponds to human insulin, wherein Y is Thr (B30); Z isArg-Arg; R¹ is Phe (B1); R³ is Gly (A21); and A2-A20 is the amino acidsequence of the A chain of human insulin (amino acid residues 2 to 20)and B2-B29 is the amino acid sequence of the B chain of human insulin(amino acid residues 2 to 29).
 16. The process according to claim 13,wherein R² is a) a hydrogen atom, or b) a peptide having 2 to 25 aminoacid residues, comprising the amino acid residue arginine (Arg) at thecarboxyl end of the peptide; R¹ is a phenylalanine residue (Phe);(B2-B29) are the amino acid residues in the positions B2 to B29 of the Bchain of human insulin; Y is an amino acid residue from the groupconsisting of alanine (Ala), threonine (Thr) and serine (Ser); X is theamino acid residue arginine (Arg) or a peptide having the amino acidsequence of the C chain of human insulin; (A2-A20) are the amino acidresidues in the positions A2 to A20 of the A chain of human insulin; andR³ is an amino acid residue from the group consisting of asparagine(Asn), serine (Ser) and glycine (Gly).
 17. The process according toclaim 13, wherein R² is a) a hydrogen atom, or b) a peptide having 2 to15 amino acid residues, at whose carboxyl end is found an arginineresidue (Arg); R¹ is a phenylalanine residue (Phe); (B2-B29) are theamino acid residues in the positions B2 to B29 of the B chain of humaninsulin; Y is a threonine residue (Thr); X is the amino acid residuearginine (Arg) or a peptide having 2 to 35 amino acid residues, where atthe beginning and at the end of the peptide there are two basic aminoacid residues, in particular arginine (Arg) and/or lysine (Lys);(A2-A20) are the amino acid residues in the positions A2 to A20 of the Achain of human insulin; and R³ is the amino acid residue asparagine(Asn) or glycine (Gly).